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解放军总医院医学创新研究部、国家老年疾病临床医学研究中心(解放军总医院)、解放军总医院第六医学中心心血管病医学部
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中华老年多器官疾病杂志编辑委员会
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E-mail: zhlndqg@mode301.cn
创刊人 王士雯
总编辑 范利
副总编辑 陈韵岱
执行主编 叶大训
编辑部主任 王雪萍
ISSN 1671-5403
CN 11-4786
创刊时间 2002年
出版周期 月刊
邮发代号 82-408
友情链接
王芳,何思毅,陈杰.Rasal2在肺动脉平滑肌细胞增殖及迁移中的作用机制[J].中华老年多器官疾病杂志,2022,21(9):684~690
Rasal2在肺动脉平滑肌细胞增殖及迁移中的作用机制
Role and mechanism of Rasal2 in proliferation and migration of pulmonary artery smooth muscle cells
投稿时间:2022-01-12  
DOI:10.11915/j.issn.1671-5403.2022.09.148
中文关键词:  Rasal2  肺动脉平滑肌细胞  增殖  迁移
英文关键词:Ras protein activator like 2  pulmonary artery smooth muscle cells  proliferation  migration This work was supported by the Project of General Hospital of Western Theater Command
基金项目:西部战区总医院院管课题(2021-XZYG-B31)
作者单位E-mail
王芳 西部战区总医院 烧伤科,成都610083 chenjie181213@sina.comrole 
何思毅 西部战区总医院 心脏外科,成都610083 chenjie181213@sina.comrole 
陈杰 西部战区总医院 心脏外科,成都610083 chenjie181213@sina.comrole 
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中文摘要:
      目的 探讨Ras蛋白激活类似物2(Rasal2)调控肺动脉平滑肌细胞(PASMCs)增殖与迁移的作用机制。方法 提取慢性-低氧小鼠肺动脉组织以及使用低氧条件刺激PASMCs,检测Rasal2表达变化。体外沉默Rasal2后检测各组哺乳动物雷帕霉素靶蛋白复合体1(mTORC1)下游效应分子S6、4EBP1的蛋白磷酸化变化,Ki-67免疫荧光染色与Transwell分析检测各组细胞增殖与迁移能力变化。使用胰岛素恢复mTORC1活性,检测各组增殖与迁移能力变化。结果 与常氧小鼠(Normoxic)相比,慢性低氧组小鼠(CH-PH)肺动脉Rasal2 mRNA[(2.57±0.15)和(1.02±0.09),P<0.001]和蛋白[(2.18±0.36)和(0.97±0.14),P<0.001]表达显著升高。与常氧组(Normoxic)细胞相比,低氧组(Hypoxic)细胞处理12、24h后Rasal2 mRNA[(2.41±0.20)、(2.86±0.24)和(1.03±0.12);均P<0.001]和蛋白[(2.50±0.32)、(2.79±0.38)和(1.13±0.12);均P<0.01]表达显著升高。与siCon转染组相比,siRasal2转染组细胞Rasal2表达显著降低[(1.09±0.09)和(0.33±0.04);P<0.001]。与Normoxic+siCon组相比,Hypoxic+siCon组Ki-67阳性细胞比例[(62.75±7.54)%和(13.90±2.35)%;P<0.001]、迁移细胞比例[(305.25±58.28)%和(88.90±13.67)%;P<0.05]、p-S6Ser235/236/S6[(3.27±0.24)和(1.06±0.14);P<0.001]、p-4EBP1Thr37/46/4EBP1[(2.95±0.15)和(1.07±0.06);P<0.001]表达均显著升高;而与Hypoxic+siCon组相比,Hypoxic+siRasal2组Ki-67阳性细胞比例[(19.25±5.34)%和(62.75±7.54)%;P<0.001]、迁移细胞比例[(115.25±19.00)%和(305.25±58.28)%;P<0.05]、p-S6Ser235/236/S6[(1.90±0.06)和(3.27±0.24);P<0.001]、p-4EBP1Thr37/46/4EBP1[(1.70±0.13)和(2.95±0.15);P<0.001]表达均显著降低。此外,与Hypoxic+siRasal2组相比,Hypoxic+siRasal2+Insulin组p-S6Ser235/236/S6[(0.94±0.16)和(0.36±0.11);P<0.01]、p-4EBP1Thr37/46/4EBP1[(0.99±0.09)和(0.61±0.03);P<0.01]表达、Ki-67阳性细胞比例[(61.25±3.10)%和(27.00±3.46)%;P<0.001]、迁移细胞比例[(315.00±21.49)%和(141.00±15.30)%;P<0.001]显著回升。结论 Rasal2可通过mTORC1途径促进PASMCs增殖及迁移。
英文摘要:
      Objective To explore the role and mechanism of Ras protein activator like 2 (Rasal2) in the proliferation and migration of pulmonary artery smooth muscle cells (PASMCs). Methods The expression of Rasal2 in pulmonary arteries of chronic hypoxia-induced pulmonary hypertension (CH-PH) mice and in hypoxia-challenged PASMCs was analyzed by Western blotting. After Rasal2 in PASMCs was silenced by transfection of siRNA, the phosphorylation levels of downstream effector molecules of mammalian target of rapamycin complex 1 (mTORC1), S6 and 4E-binding protein 1 (4EBP1) were determined by Western blotting, and the proliferation and migration of PASMCs were measured with Ki-67 immunofluorescence staining and Transwell assay, respectively. The proliferation and migration of PASMCs were further determined after restoration of mTORC1 activity by insulin. Results Compared with the Normoxic group, the mRNA [(2.57±0.15) vs (1.02±0.09); P<0.001) and protein [(2.18±0.36) vs (0.97±0.14); P<0.001) levels of Rasal2 were significantly increased in the pulmonary artery of the CH-PH group. In the PASMCs of the Hypoxic group, the mRNA [(2.41±0.20), (2.86±0.24) vs (1.03±0.12); both P<0.001] and protein [(2.50±0.32), (2.79±0.38) vs (1.13±0.12); both P<0.01] levels of Rasal2 were obviously higher after 12 h and 24 h of exposure when compared with the Normoxic group. Compared with the siCon group, the protein level of Rasal2 in PASMCs of the siRasal2 group was significantly deceased [(1.09±0.09) vs (0.33±0.04), P<0.001]. Compared with the Normoxic+siCon group, the percentage of Ki-67-positive cells [(62.75±7.54)% vs (13.90±2.35)%; P<0.001], percentage of migrated cells [(305.25±58.28)% vs (88.90±13.67)%; P<0.05], and expression of p-S6Ser235/236/S6 [(3.27±0.24) vs (1.06±0.14); P<0.001] and p-4EBP1Thr37/46/4EBP1 [(2.95±0.15) vs (1.07±0.06); P<0.001] were both increased in the Hypoxic+siCon group. Compared with the Hypoxic+siCon group, the percentage of Ki-67-positive cells [(19.25±5.34)% vs (62.75±7.54)%; P<0.001], percentage of migrated cells [(115.25±19.00)% vs (305.25±58.28)%; P<0.05], and expression of p-S6Ser235/236/S6 [(1.90±0.06) vs (3.27±0.24); P<0.001] and p-4EBP1Thr37/46/4EBP1 [(1.70±0.13) vs (2.95±0.15); P<0.001] in the Hypoxic+siRasal2 group were both reduced. Additionally, the percentage of Ki-67-positive cells [(61.25±3.10)% vs (27.00±3.46)%; P<0.001], percentage of migrated cells [(315.00±21.49)% vs (141.00±15.30)%; P<0.001], and expression of p-S6Ser235/236/S6 [(0.94±0.16) vs (0.36±0.11); P<0.01] and p-4EBP1Thr37/46/4EBP1 [(0.99±0.09) vs (0.61±0.03); P<0.01] were both elevated in the Hypoxic+siRasal2+Insulin group than the Hypoxic+siRasal2 group. Conclusion Rasal2 promotes the proliferation and migration of PASMCs via mTORC1 pathway.
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